4′-C-[(4-trifluoromethyl-1 H -1,2,3-triazol-1-yl)methyl]thymidine as a sensitive 19F NMR sensor for the detection of oligonucleotide secondary structures

Article abstract of DOI:10.1021/jo500326j

4′-C-[(4-Trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]thymidine was synthesized and incorporated as a phosphoramidite into oligonucleotide sequences. Its applicability as a sensor for the ¹⁹F NMR spectroscopic detection of DNA and RNA secondary structures was demonstrated. On DNA, the ¹⁹F NMR measurements were focused on monitoring of duplex-triplex conversion, for which this fluorine-labeled 2′- deoxynucleoside proved to be a powerful sensor. This sensor seemingly favors DNA, but its behavior in the RNA environment also turned out to be informative. As a demonstration, invasion of a 2′-O-methyl oligoribonucleotide into an RNA hairpin model (HIV-1 TAR) was monitored by ¹⁹F NMR spectroscopy. According to the thermal denaturation studies by UV spectroscopy, the effect of the 4′-C-(4-trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl moiety on the stability of these DNA and RNA models was marginal.

Full text of DOI:10.1021/jo500326j

Article
pubs.acs.org/joc
4′‑C‑[(4-Trifluoromethyl‑1H‑1,2,3-triazol-1-yl)methyl]thymidine as a
19
Sensitive F NMR Sensor for the Detection of Oligonucleotide
Secondary Structures
Lotta Granqvist and Pasi Virta*
Department of Chemistry, University of Turku, 20014 Turku, Finland
S
* Supporting Information
ABSTRACT: 4′-C-[(4-Trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]-
thymidine was synthesized and incorporated as a phosphoramidite into
oligonucleotide sequences. Its applicability as a sensor for the 19F NMR
spectroscopic detection of DNA and RNA secondary structures was
demonstrated. On DNA, the 19F NMR measurements were focused on
monitoring of duplex−triplex conversion, for which this fluorine-labeled 2′-
deoxynucleoside proved to be a powerful sensor. This sensor seemingly
favors DNA, but its behavior in the RNA environment also turned out to
be informative. As a demonstration, invasion of a 2′-O-methyl oligoribonucleotide into an RNA hairpin model (HIV-1 TAR) was
monitored by 19F NMR spectroscopy. According to the thermal denaturation studies by UV spectroscopy, the effect of the 4′-C-
(4-trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl moiety on the stability of these DNA and RNA models was marginal.
INTRODUCTION
■
Previous studies of 19F NMR spectroscopy have shown it to be
a promising tool for the characterization of molecular
interactions, secondary structural arrangements, and their
dynamics in oligonucleotides.1−19 The sensitivity is still far
from that of the conventional spectrophotometric measure-
ments (UV, CD, and fluorescence-based techniques), but 19F
NMR may give more detailed information about the conversion
mechanisms and relative mole fractions of the secondary
structural species. The efficient discrimination even between
only moderately different structures results from the wide
chemical shift dispersion of the 19F nucleus (50-fold compared
to 1H) and from the characteristic shift, which responds readily
to changes in local van der Waals interactions and electrostatic
fields.19−23 It may even be suggested that once an appropriate
label is readily available and its incorporation into oligonucleo-
tides is facile, 19F NMR measurements could be one of the
routine methods for the characterization of molecular
interactions and competing hybridization modes. In order to
increase the sensitivity (83% of 1H NMR sensitivity) and
straightforwardness of the 19F NMR measurement, there is an
interest in developing nucleoside derivatives to which magneti-
cally equivalent fluorine atoms (in a CF3 group) are attached
via an appropriate proton-coupling barrier [i.e., an isolated spin
system provided by alkyne, thioether, or aryl bridges (cf. Figure
1); precisely, the CF3 fluorines are magnetically equivalent only
if their rotation rate is much faster than the effective correlation
time of the nucleic acid structure]. These 19F-multiplied sensors
may allow rapid measurements at micromolar oligonucleotide
concentrations without the need for 1H decoupling techniques.
The following obvious demands, which more or less
compromise each other, should additionally be considered
with the sensors: the effect on the native oligonucleotide
Figure 1. High-performance 19F multiplied NMR sensors used for
oligonucleotides.
structure should be marginal, while the resulting shift should
clearly reflect the secondary structural changes. Examples of
previously reported 19F-multiplied sensors are presented in
Figure 1. 5-[4,4,4-Trifluoro-3,3-bis(trifluoromethyl)but-1-ynyl]-
2′-deoxyuridine (1), with nine equivalent fluorine atoms, has
successfully been used for the detection of DNA and RNA
double helices.7,9,12 Although notable effects on the stabilities of
the labeled DNA and RNA structures have not been observed,
the bulky 4,4,4-trifluoro-3,3-bis(trifluoromethyl)but-1-ynyl
group undeniably plays a dominating role in the major groove.
2′-Deoxy-5-(trifluoromethyl)uridine (2) is a commercially
Received: February 11, 2014
Published: March 28, 2014
© 2014 American Chemical Society
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Figure 2. 19F NMR signals of sensor 6 in different DNA hybrization modes. Conditions: (i) 100 μmol L−1 ON1, 10 mmol L−1 sodium phosphate
(pH 5.5), 2 mmol L−1 MgCl2, and 0.1 mol L−1 NaCl in D2O−H2O (1:9 v/v) at 25 °C; (ii) the same as (i) except at pH 6.0 and 37 °C; (iii) 50 μmol
L
−1 ON1, 45 μmol L−1 ON2, 10 mmol L−1 sodium phosphate (pH 6.0), 2 mmol L−1 MgCl2, and 0.1 mol L−1 NaCl in D2O−H2O (1:9 v/v) at 37
°C; (iv) 50 μmol L−1 ON1, 50 μmol L−1 ON2, 50 μmol L−1 ON3, 10 mmol L−1 sodium phosphate (pH 6.0), 2 mmol L−1 MgCl2, and 0.1 mol L−1
NaCl in D2O−H2O (1:9 v/v) at 50 °C, with the passive temperature-dependent shift eliminated and the shifts interpolated to that at 37 °C; (v) 100
μmol L−1 ON1, 100 μmol L−1 ON3, 100 μmol L−1 ON4, 10 mmol L−1 sodium phosphate (pH 6.0), 2 mmol L−1 MgCl2, 0.1 mol L−1 NaCl in D2O−
H2O (1:9 v/v) at 37 °C. (Note: in spite of the virtual homogeneity of the oligonucleotides, the shoulders of the 19F NMR signals most likely
originate from impurities.)
19F NMR spectroscopic characterization of RNA invasion9 was
available nucleoside that has been incorporated into DNA
double helices.24 In spite of this modest base modification, a
repeated and confirmed. The 4-trifluoromethyl-1H-1,2,3-
remarkable decrease in the duplex stabilities has been
observed.17,24 Additionally, this label is not compatible with
the standard oligonucleotide protection scheme, since it is
readily transformed to a 5-cyanouridine residue upon
ammonolysis.24 19F NMR applications with this potential
sensor have not been described. Recently, 3,5-bis-
(trifluoromethyl)benzoic acid (3) and 4-[3,5-bis-
(trifluoromethyl)benzamido]benzoic acid (4) were postsyn-
thetically coupled to 5-(3-aminopropyn-1-yl)-2′-deoxyuridine
residues of oligonucleotide sequences, and the resulted sensors
were used to probe mismatched and bulged DNAs by 19F NMR
spectroscopy.18 Comparable to 1, these sterically demanding
major-groove-oriented base modifications do not remarkably
retard hybridization with complementary oligonucleotides.
Elegant 19F NMR applications (structure probing of bistable
RNA, characterization of RNA−protein and RNA−small-
molecule interactions) using 2′-deoxy-2′-trifluoromethylthiour-
idine (5) as a sensor have been described.16 The synthesis of
phosphoramidite derivative 5 and its incorporation into an
RNA sequence were straightforward, and the resulting 19F
NMR shift of the sensor clearly distinguished between different
secondary structures. With this sensor, however, a clearly
facilitated thermal denaturation (57 vs 72 °C) of an RNA
hairpin was reported.
triazol-1-yl group offered a quasi-isolated spin system in a
similar manner as a trifluoromethylphenyl group (cf. 3 and 4),
and 19F NMR data could be obtained without the need for
fluorine−proton decoupling. The effect of sensor 6 on the
stability of these DNA and RNA models was marginal, although
it expectedly depended slightly on the labeling site (Table 1).
Table 1. UV Melting Temperatures (Tm) of the 19F-Labeled
Secondary Structures of Oligonucleotides (X = 6) and
Values of ΔTm in Comparison with Unmodified
a
Oligonucleotides (X = Thymidine or Uridine)
entry
oligonucleotides labeled with 6
Tm/°C (ΔTm/°C)
1
2
3
4
5
6
ON1/ON2 duplex
54.9 (−0.6)
32.9 (−0.6)
31.0 (−1.7)
72.4 (+0.5)
71.0 (−0.9)
69.2 (−2.7)
ON1/ON2/ON3 triplex
ON1/ON4/ON5 triplex
19F-HIV-1 TAR(1)
19F-HIV-1 TAR(2)
19F-HIV-1 TAR(3)
a
Conditions: 10 mmol L−1 NaH2PO4 (pH 6.0), 0.1 mol L−1 NaCl, 2.0
mmol L−1 MgCl2, and 2.0 μmol L−1 each oligonucleotide (entries 1−
3); 10 mmol L−1 sodium cacodylate (pH 7.0), 0.1 mol L−1 NaCl, and
2.0 μmol L−1 each oligonucleotide (entries 4−6).
In response to the above demands, a novel 4′-C-modified 19
F
RESULTS AND DISCUSSION
NMR sensor, 4′-C-[(4-trifluoromethyl-1H-1,2,3-triazol-1-yl)-
methyl]thymidine (6), is described in the present report.
This sensor (as a deoxynucleoside with predominant S
conformation) was primarily designed for the detection of
■
4′-C-[(4-Trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]-
thymidine Sensor 6 and Synthesis of Its Phosphor-
amidite Derivative 12. The 4′-C is an attractive site for the
introduction of a label in DNA. Previous studies have shown
that the 4′-modification faces the minor groove upon
hybridization, and oligonucleotides modified at this site usually
form equally stable double helices compared with their
unmodified counterparts.25−34 It may also be expected that
the fluorine label at this site in 6 only marginally disturbs the
secondary structure of DNA but is sensitive to secondary
structural arrangements because of the particular orientation.
The 4′-C additionally is a uniform site for the label compared
with, for example, the base-modified probes 1−4. On RNA,
DNA secondary structures, which was demonstrated by the 19
F
NMR spectroscopic monitoring of DNA triplex/duplex/single
strand conversion. For these experiments 6 proved to be an
excellent sensor, resulting in sharp and well-distinguished 19F
signals as unique singlets for DNA triplexes (6 paired via both
the Watson−Crick face and the Hoogsteen face), a DNA
duplex, and a single strand (Figure 2). 6 turned out to be
informative also for the RNA environment. Three different sites
of an HIV-1 trans-activation response element (TAR) RNA
model were labeled by 6, and our previously reported study of
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however, certain shortcomings of 6 may be expected: 6 lacks
the 2′-OH group, and the predominant S conformation may
disfavor A-type helices. Additionally, on an RNA duplex the (4-
trifluoromethyltriazolyl)methyl group at the 4′-C position is
oriented outward from the helix, not onto the edge of the
minor groove as in DNA, and hence, only modest 19F NMR
spectroscopic discrimination between double-stranded and
single-stranded RNA may be expected. In spite of these
presumptions, the behavior of 6 in the RNA environment
proved promising (Table 1; also see Figures 5 and 6 below).
The 4′-trifluoromethyl-1H-1,2,3-triazol-1-yl moiety was chosen
because it may be readily obtained from the corresponding
azide derivative 1033 (note: the triazolyl moiety is a weak
base,35 which is entirely uncharged in the 19F NMR
measurements used for the oligonucleotides below) via
stereocontrol for the 4′-C substituent is facile only in
thymidine. The anhydronucleoside 940 determined the stereo-
control.
Oligonucleotide Synthesis. Oligonucleotides were syn-
thesized on a 1.0 μmol scale using an automatic DNA/RNA
synthesizer. Benzylthiotetrazole as an activator and coupling
times of 20 and 300 s were used for the couplings of standard
DNA and RNA building blocks, respectively. For the fluorine-
labeled thymidine phosphoramidite 12 (using a 0.13 mol L−1
solution in dry MeCN to load the reagent vessel), the coupling
time was increased to 600 s, resulting in a coupling efficiency of
92%. Nearly quantitative coupling of 12 could be provided by
repeating the automatic coupling step or by using a manual
phosphoramidite coupling in which a more concentrated
solution (final concentration 0.11 mol L−1 after addition of
the activator) but a smaller excess (10 equiv) of 12 could be
used (see the Experimental Procedures). Otherwise, standard
DNA and RNA protocols were applied. The oligonucleotides
were released from the support with concentrated ammonia
(33% aqueous NH3 at 55 °C overnight), and TBDMS
protections were removed by treatment with triethylamine
trihydrofluoride followed by filtration through an ion exchange
cartridge. The purification was carried out by RP HPLC.
According to the RP HPLC profiles (see the Supporting
Information), only a slight decrease in the purities of the
labeled sequences compared with that of the unlabeled
sequence was observed. The authenticity of the oligonucleo-
tides was verified by MS (ESI-TOF).
copper(I)-catalyzed Huisgen 1,3-dipolar cycloaddition.36,37
A
high-yielding procedure for the preparation of 4′-C-azidometh-
yl-3′-O-(4,4′-dimethoxytrityl)thymidine (10) (Scheme 1) was
a
Scheme 1. Synthesis of Phosphoramidite Building Block 12
19F NMR Measurements. Instead of testing probe 6 for
new, sophisticated 19F NMR applications, its behavior was
demonstrated in relatively well studied and defined secondary
structures: in a DNA duplex, in DNA triplexes (6 paired via
both the Hoogsteen face and the Watson−Crick face), and in
HIV-1 TAR models upon invasion with 2-O-methyl oligor-
ibonucleotides in which three different sites were labeled: (1)
the stem region before the bulge, (2) the stem region between
the loop and the bulge, and (3) the loop. In this manner we
also could get rather comprehensive understanding of how this
sensor affects the stabilities of native oligonucleotide structures
in varying local environments of the sensor (Table 1).
As a first example, we repeated the experiment previously
reported by Tanabe et al.10 They used 5-fluoro-2′-deoxyuridine
as an 19F signal transmitter for the detection of triplex
formation between ON1, ON4, and ON5 (Figure 3). In
contrast to their experiments, lower oligonucleotide concen-
trations of 50 and 100 μmol L−1 (vs 250 μmol L−1) and a
higher pH of 6.0 (vs 5.5) were used for the triplex formation. At
25 °C and pH 5.5, ON1 (100 μmol L−1, X = 4′-C-[(4-
trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]thymidine, 10
mmol L−1 sodium phosphate, 2 mmol L−1 MgCl2, 0.1 mol
a
Conditions: (i) 3,3,3-trifluoroprop-1-yne, CuSO4, sodium ascorbate;
(ii) 2-cyanoethyl N,N-diisopropylphosphoramidochloridite, Et3N,
DCM.
previously reported.33,38 Accordingly, 4′-C-hydroxymethylthy-
midine 8 was first synthesized from thymidine (7) following the
literature procedure.38,39 The primary hydroxyl groups were
converted to triflates and then the O2,5′-anhydronucleoside was
formed,40 after which the remaining 4′-C-trifluoromethanesul-
fonyl group was replaced with azide ion to give 9. The
anhydronucleoside bridge was hydrolyzed, the 3′-OH group
exposed, and the 5′-OH group protected with a DMTr group.
Several synthetic steps were required, but the procedure was
reproducible and the overall yield from 7 to 10 was relatively
high (49%). Copper(I)-catalyzed Huisgen 1,3-dipolar cyclo-
addition36 between the 4′-C-azidomethyl group of 10 and
gaseous 3,3,3-trifluorobutyne gave 11 with the trifluoromethyl-
triazolyl moiety in 77% yield, and the 3′-OH group was then
quantitatively (97%) phosphitylated to afford the desired
phosphoramidite 12. The above-described synthesis of 4′-C
modification evidently may be generalized for other nucleo-
sides, but the fact should be borne in mind that the correct
L
−1 NaCl) appeared as two signals in the 19F NMR spectrum (i
in Figure 2), as described by Tanabe et al.10 The sharp signal at
−62.53 ppm referred to the single strand, and the broad signal
ca. 0.6 ppm upfield resulted from unspecific C−CH+ quartets
(but not necessarily from a specific i motif). At a higher pH of
6.0 (at 37 °C), only the sharp signal at −62.53 ppm was
detected (ii in Figure 2). The triplex formation was then carried
out (I in Figure 3). ON1 was titrated with ON4/ON5 duplex,
which should result in binding of ON1 via the Hoogsteen face.
As shown in I in Figure 3, clear conversion of the initial signal
at −62.53 ppm to a new sharp signal at −61.69 ppm occurred,
which indicated ON1/ON4/ON5 triplex formation. Since
sharp and well-distinguished signals (Δδ = 0.83 ppm) were
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Figure 3. (I) Formation and (II, III) thermal melting of the ON1/ON4/ON5 DNA triplex followed by 19F NMR spectroscopy. Conditions: 100
μmol L−1 ON1, 0−120 μmol L−1 ON4 and ON5 (1:1 mol/mol), 10 mmol L−1 sodium phosphate (pH 6.0), 2 mmol L−1 MgCl2, and 0.1 mol L−1
NaCl in D2O−H2O (1:9 v/v).
Figure 4. DNA triplex/duplex/single strand conversions followed by 19F NMR spectroscopy. Conditions: 50 μmol L−1 ON1, ON2, and ON3, 10
mmol L−1 sodium phosphate (pH 6.0), 2 mmol L−1 MgCl2, and 0.1 mol L−1 NaCl in D2O−H2O (1:9 v/v).
detected, we next studied whether the relative peak areas at
increasing temperature (II in Figure 3) could be applied to the
determination of the melting temperature (Tm) of the ON1/
ON4/ON5 triplex. As shown, a nice negative S curve was
obtained (III in Figure 3), in which the inflection point at 66
°C showed thermal melting. [The difference between the Tm
values in 100 μmol L−1 (66 °C) and 2 μmol L−1 (31.0 °C)
solutions of oligonucleotides should be noted (Table 1)].
The 19F NMR spectroscopic behavior of the sensor was then
similarly studied upon formation of the ON1/ON2 duplex and
the ON1/ON2/ON3 triplex. Sharp and unique signals resulted
also for these secondary structures at −62.17 and −61.86 ppm,
respectively (Figure 2; the more detailed data of the titrations
are not presented). Melting of the ON1/ON2/ON3 triplex
bearing the sensor in the Watson−Crick face was more
informative than that of ON1/ON4/ON5 triplex (Figure 4).
As shown, the conversion from the ON1/ON2/ON3 triplex to
the ON1/ON2 duplex and finally to the ON1 single strand
could be nicely followed according to the relative peak areas of
the 19F NMR resonance signals. The oligonucleotide
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Figure 5. 19F NMR shift-vs-temperature profiles of 19F-labeled HIV-TAR models and their invasion complexes. Conditions: 50 μmol L−1 labeled
19F-HIV-1 TAR(1−3) with and without 50 μmol L−1 ON6, 10 mmol L−1 sodium cacodylate (pH 7.0), and 0.1 mol L−1 NaCl in D2O−H2O (1:9 v/
v).
concentration in this experiment was 50 μmol L−1. It may be
worth mentioning that a seemingly direct conversion of the
ON1/ON2/ON3 triplex to ON1 was observed at a
concentration of 100 μmol L−1 (in our initial attempt), which
was a result of the stronger concentration dependence of the
triplex compared with the duplex.
A/B and C/D equilibria were observed, which indicated that
rearrangements between these structures were slow on the
NMR time scale. For intramolecular hybridizations, the
equilibrium is usually fast on the NMR time scale,6 which
questions our previous conclusions.
In the present study, 6 was incorporated at three different
positions of the same HIV-1 TAR model (X1, X2, and X3 in 19F-
HIV-1 TAR(1), 19F-HIV-1 TAR(2) and 19F-HIV-1 TAR(3),
respectively; Figure 5), and the RNA invasion studies were
repeated. 19F NMR measurements were carried out using
oligonucleotide concentrations of 50 μmol L−1 in 0.10 mol L−1
NaCl buffered by 10 mmol L−1 sodium cacodylate, pH 7.0
(previous conditions: 20 μmol L−1 19F-labeled HIV-1 TAR with
ON6 in 0.1 mol L−1 NaCl with 25 mmol L−1 NaH2PO4, pH
6.5). The thermal UV melting temperatures for the HIV-1 TAR
models under these experimental conditions (69.2−72.4 °C)
are listed in Table 1. The 19F NMR spectra of each labeled
HIV-1 TAR model with and without 1.0 equiv of ON6 were
measured at varying temperatures. The 19F NMR shift-versus-
temperature profiles are presented in I in Figure 5, and the
profiles of the shift differences versus temperature after
reduction of the passive temperature-dependent shift are
shown in II in Figure 5. The thermal melting of HIV-1 TAR
(A/B equilibrium) could be monitored by the stem-located
sensors (X2 and X3) as negative S curves (■, ●), while 6 in the
loop (X1) shifted continuously (◀ over 0.5 ppm) over the
whole temperature range measured (23−75 °C). In comparison
to the UV melting profiles (Table 1), slightly different Tm
values were obtained from the 19F NMR shift-versus-temper-
ature profiles, given by the inflection points at 71 °C for 19F-
HIV-1 TAR(2) and at 68 °C for 19F-HIV-1 TAR(3) (II in
Figure 5). It is worth noting that now well-behaving
coalescence signals were obtained [see the spectra of 19F−
Triple helices may be stabilized by aminoglycosides.41−43
Among them, neomycin has proven to be the most effective
ligand. The above-described triplex/duplex/single strand
conversion was followed also in the presence of neomycin
(Figure 4, blue curves). As shown, 1.0 equiv of neomycin was
enough to stabilize the ON1/ON2/ON3 triplex (50 μmol L−1)
clearly (ΔTm = 4 °C), whereas the ON1/ON2 duplex was less
stabilized. The difference between these stabilizations was
consistent with the literature.41−43 Oligonucleotides have been
conjugated to aminoglycosides in order to increase their
affinities to RNA targets,12,33,34,44,45 but attempts to use them
for triplex recognition have failed to date. However, the
approach may still have some potential, since the observed
enhanced triplex formation shown in Figure 4 resulted from
only a stoichiometric amount of neomycin.
We previously used 19F NMR spectroscopy to characterize
the invasion of 2′-O-methyl oligoribonucleotides to an 19F-
labeled HIV-1 TAR model,9 and later the same experiment was
applied for the verification of aminoglycoside-enhanced
invasion.12 In these previous reports, sensor 17 was
incorporated into the tetranucleotide stem region between
the bulge and the loop of an HIV-1 TAR model [cf. 19F-HIV-1
TAR(2) in Figure 5 with X2 = 1 and X1 = X3 = U], and it was
able to detect not only the denaturation of HIV-1 TAR but also
the conversion of the macro-looped complex to the open-chain
invasion complex (cf. structures A, B, C, and D in Figure 5).
Interestingly, separate, albeit partially coalescent, signals for the
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Figure 6. 19F NMR spectra of 19F-labeled HIV-TAR models and their invasion complexes in increasing temperature. Conditions: 50 μmol L−1
labeled 19F-HIV-1 TAR(1−3) with and without 50 μmol L−1 ON6 (titration carried out in II), 10 mmol L−1 sodium cacodylate (pH 7.0), and 0.1
mol L−1 NaCl in D2O−H2O (1:9 v/v) (cf. the shift data in Figure 5).
HIV-1 TAR(2) in panel I of Figure 6]. Thus, it seems that a
heavily modified sensor such as 1, even if it does not decrease
the stability of the secondary structure, may misrepresent the
rate of denaturation. The equilibrium between the macro-loop
invasion complex (C) and the open-chain invasion complex
(D) could be monitored by labeled 19F-HIV-1 TAR(3) (X3 =
6, X1 = X2 = U). Also here, an averaged signal between the
structures was obtained, but the signal was broad (III in Figure
6). The incomplete coalescence signal refers to a slower process
in comparison with the hairpin melting (A/B). The inflection
point at 39 °C in the shift-versus-temperature profile (▼),
corresponding to Tm of the macro-loop complex C, is clearly
seen. 6 in 19F-HIV-1 TAR(2) (X2 = 6, X1 = X3 = U) also
reflected the C/D equilibrium, but an unclear shift-versus-
temperature profile was obtained (▲). As shown in II in
Figure 5, the signals of the hairpin HIV-1 TAR (A) and the
macro-loop complex C overlapped, but the titration with ON6
could be followed at 55 °C (i.e., under conditions where A was
directly converted to the open-chain invasion complex D; II in
Figure 6). Finally, melting of the open-chain invasion complex
D to give the denatured HIV-1 TAR (B) could be followed by
relative 19F NMR peak areas using 19F-HIV-1 TAR(1) (X1 = 6,
X2 = X3 = U) (IV in Figure 6). As shown by these results, the
19F shift difference between the signals of the single-stranded
and double-stranded RNA was only slightly smaller than that
with DNA, and invasion of a 2′-O-methyl oligoribonucleotide
into a HIV-1 TAR model and its mechanism could be clearly
characterized by 19F NMR spectroscopy using 6 as a signal
transmitter (Figures 5 and 6).
CONCLUSION
■
A new 19F-multiplied sensor for the 19F NMR spectroscopic
detection of oligonucleotide secondary structures, 4′-C-[(4-
trifluoromethyl-1H-1,2,3-triazol-1-yl)methyl]thymidine (6), has
been described. The synthesis of the corresponding phosphor-
amidite building block 12 was straightforward, and it was
readily incorporated into DNA and RNA sequences by an
automatic synthesizer. The uniform 4′-C position for the
fluorine sensor proved to behave nicely in DNA and RNA
environments. In 19F NMR spectroscopy, 6 readily reflected
secondary structural arrangements but only slightly affected the
stabilities compared with the native oligonucleotide structures
(as verified by UV melting profiles). 19F NMR spectroscopic
characterization of DNA triplex/duplex/single strand con-
version and of invasion of an RNA hairpin model (HIV-1 TAR)
was demonstrated.
EXPERIMENTAL PROCEDURES
General Remarks. Dichloromethane and MeCN were dried over 4
■
Å molecular sieves, and triethylamine was dried over CaH2. NMR
spectra were recorded at 500 MHz. The chemical shifts for 1H and 13
C
NMR are given in parts per million from the residual signals of the
deuterated solvents (CD3OD and CD3CN). 31P NMR shifts are
referenced to external H3PO4 and 19F NMR shifts to external CCl3F.
Mass spectra were recorded using electrospray ionization (ESI).
5′-O-(4,4′-Dimethoxytrityl)-4′-C-[(4-trifluoromethyl-1H-
1,2,3-triazol-1-yl)methyl]thymidine (11). To a mixture of 4′-C-
azidomethyl-5′-O-(4,4′-dimethoxytrityl)thymidine (10)33 (0.86 g, 1.4
mmol) in DMSO (6 mL) were added aqueous solutions of CuSO4
(0.1 mol L−1, 150 μL, 15 μmol) and sodium ascorbate (0.1 mol L−1,
1.5 mL, 0.15 mmol). The reaction was carried out in a sealed tube
placed in an ice bath and bubbled with gaseous 3,3,3-trifluoropropyne
for 5 min. The reaction mixture was then stirred at room temperature
for 48 h, during which time the gas was added three times in an ice
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bath along with additions of the aqueous solutions of CuSO4 (100 μL,
10 μmol) and sodium ascorbate (400 μL, 40 μmol). The reaction
mixture was then partitioned between dichloromethane and water.
The organic phase was separated, dried over Na2SO4, and evaporated
to dryness. The residue was purified by silica gel chromatography
(30−0% hexane and 0.1% Et3N in EtOAc) to yield 0.77 g (77% yield)
of 11 as a yellowish oil. 1H NMR (500 MHz, CD3OD) δ: 8.38 (s, 1H),
7.43 (s, 1H), 7.40−7.38 (m, 2H), 7.28−7.19 (m, 7H), 6.82−6.81 (m,
4H), 6.43 (dd, 1H, J = 7.3 and 6.1 Hz), 4.88 (d, 1H, J = 14.7 Hz),
4.79−4.76 (m, 2H), 3.74 (s, 6H), 3.25 (d, 1H, J = 10.3 Hz), 2.87 (d,
1H, J = 10.3 Hz), 2.44 (m, 1H), 2.33 (ddd, 1H, J = 13.7, 6.1, and 3.1
Hz), 1.42 (s, 3H). 13C NMR (125 MHz, CD3OD) δ: 164.8, 159.0,
158.9, 150.9, 144.3, 137.8 (q, J = 39.0 Hz), 136.1, 135.0, 134.7, 130.0,
127.9, 127.6, 126.8, 125.9, 120.7 (q, J = 266.7 Hz), 112.8, 110.6, 87.3,
86.4, 84.5, 72.1, 64.1, 54.3, 52.0, 39.6, 10.7. 19F NMR (470 MHz,
CD3OD) δ: −63.64. HRMS (ESI-TOF) m/z: calcd for
C35H34F3N5NaO7 [M + Na]+ 716.2308, found 716.2310.
assay, nearly quantitative coupling of 12 was provided. After the chain
elongation, the supports were suspended in concentrated ammonia
(33% aqueous NH3 at 55 °C overnight), the mixtures were filtered,
and the filtrates were evaporated to dryness. The residues of
oligodeoxyribonucleotides ON1−ON5 and 2′-O-methyl oligoribonu-
cleotide ON6 were dissolved in water and subjected to RP HPLC
purification. The residues of HIV-1-TAR models were dissolved in
triethylamine trihydrofluoride (75 μL), triethylamine (60 μL), and
DMSO (115 μL), mixed for 2.5 h at 65 °C, and filtered through an ion
exchange cartridge, after which the filtrates were evaporated to
dryness. The residues of the free RNAs were dissolved in sterilized
water, and then RP HPLC was carried out. After RP HPLC
purification using a semipreparative RP HPLC column (C-18, 250
mm × 10 mm, 5 μm) with gradient elution (0−90% acetonitrile in 0.1
mol L−1 triethylammonium acetate in 25 min) and detection at 260
nm, the homogenized oligonucleotides were lyophilized, and their
authenticities were verified by MS (ESI-TOF) (Table 2).
3′-O-[(2-Cyanoethoxy)-(N,N-diisopropylamino)phosphinyl]-
5′-O-(4,4′-dimethoxytrityl)-4′-C-[(4-trifluoromethyl-1H-1,2,3-
triazol-1-yl)methyl]thymidine (12). Compound 11 (0.15 g, 0.22
mmol) was dried over P2O5 in a vacuum desiccator and dissolved in
dry dichloromethane (1.5 mL). Triethylamine (152 μL, 1.1 mmol)
and 2-cyanoethyl N,N-diisopropylphosphoramidochloridite (78 μL,
0.35 mmol) were added, and the mixture was stirred under nitrogen
for 2 h. Upon completion of the reaction, the reaction mixture was
eluted through a short dried silica gel column (60−0% hexane and
0.1% Et3N in EtOAc) to yield two diastereomers of 12 as white foams
(overall 0.19 g, 97% yield). Diastereomer I: 1H NMR (500 MHz,
CD3CN) δ: 9.36 (b, 1H), 8.22 (s, 1H), 7.45−7.25 (m, 10H), 6.88 (m,
Table 2. Observed and Calculated Molecular Masses of the
Oligonucleotides Labeled with 6
observed molecular
mass
calculated average
molecular mass
entry oligonucleotide
a
1
2
ON1
4514.8
4515.0
9494.1
b
19F-HIV-1
9492.5
TAR1
b
3
4
19F-HIV-1
TAR2
9492.3
9494.1
9494.1
b
19F-HIV-1
9492.3
3
2
TAR3
4H), 6.34 (dd, J = 6.7 Hz average), 5.01 (m, 1H), 4.85 (d, 1H, J =
2
14.9 Hz), 4.74 (d, 1H, J = 14.9 Hz), 3.87−3.76 (m, 2H), 3.79 and
a
Calculated from the most intense isotope combination at [(M −
3.78 (both s, 6H), 3.73−3.66 (m, 2H), 3.24 (d, 1H, 2J = 10.4 Hz), 2.93
(d, 1H, 2J = 10.4 Hz), 2.63 (m, 2H), 2.47−2.37 (m, 2H), 1.52
(segmented, 3H), 1.25−1.23 (m, 12H). 13C NMR (125 MHz,
CD3CN) δ: 163.7, 158.9, 150.3, 144.5, 137.5 (q, J = 38 Hz), 135.8,
135.2, 135.0, 130.1, 128.0, 127.1, 126.0, 120.9 (q, J = 265 Hz), 118.4,
113.2, 110.5, 87.0, (85.79 and 85.75), 84.2, (74.2 and 74.0), 63.4, (58.8
and 58.6), 54.9, 52.2, (43.2 and 43.1), 38.2, (24.97 and 23.91), (20.1
and 20.0), 11.2. 31P NMR (200 MHz, CD3CN) δ: 149.8. 19F NMR
(470 MHz, CD3CN) δ: −63.28. Diastereomer II: 1H NMR (500 MHz,
CD3CN) δ: 9.38 (b, 1H), 8.19 (s, 1H), 7.45−7.18 (m, 10H), 6.87 (m,
b
3H)]3−/3. Calculated from the most intense isotope combination at
[(M − 11H)]11−/11.
19F NMR Spectroscopy Studies. Oligonucleotides (as triethy-
lammonium salts) were dissolved in an appropriate buffer [500 μL of
either 10 mmol L−1 sodium phosphate, 2 mmol L−1 MgCl2, 0.1 mol
L−1 NaCl in D2O−H2O (1:9 v/v), pH 5.5; the same but at pH 6.0; or
10 mmol L−1 sodium cacodylate, 0.1 mol L−1 NaCl in D2O−H2O (1:9
v/v), pH 7.0]. All of the samples were heated to 90 °C and then
allowed to cool to room temperature, after which the NMR
measurements were carried out at target temperatures. Spectra were
recorded at a frequency of 470.6 MHz on a Bruker Avance 500 MHz
spectrometer. Typical experimental parameters were chosen as
follows: 19F excitation pulse, 4.0 μs; acquisition time, 1.17 s; prescan
delay, 6.0 μs; relaxation delay, 0.8 s; usual number of scans, 2048 or
1024. The parameters were optimized to gain the signals with the
longest relaxation rate (triplex). In order to improve the authenticity of
the relative peak areas (spectra in Figures 3 and 4), the decrease
(triplexes) and increase (single strand) in the peak areas were
additionally referred to an internal standard (5-[4,4,4-trifluoro-3,3-
bis(trifluoromethyl)but-1-ynyl]uridine)12 as far as it was possible. A
macro command was used for automatic temperature ramps using a 20
min equilibration time for each temperature.
Melting Temperature Studies. The melting curves (absorbance
vs temperature) were measured at 260 nm on a PerkinElmer Lambda
35 UV−vis spectrometer equipped with a multiple cell holder and a
Peltier temperature controller. The temperature was changed from 10
to 90 °C at a rate of 0.5 °C/min. The measurements were performed
in 10 mmol L−1 sodium phosphate buffer (pH 6) containing 0.1 mol
L−1 NaCl and 2 mmol L−1 MgCl2 or in 10 mmol L−1 sodium
cacodylate (pH 7) containing 0.1 mol L−1 NaCl. The oligonucleotides
were used at a concentration of 2 μmol L−1. Each Tm value was
determined as the maximum of the first derivative of the melting curve.
3
2
4H), 6.37 (dd, J = 7.0 and 6.5 Hz), 4.96 (m, 1H), 4.80 (d, 1H, J =
15.0 Hz), 4.74 (d, 1H, 2J = 15.0 Hz), 3.97−3.75 (m, 2H), 3.78 (s, 6H),
3.74−3.63 (m, 2H), 3.15 (d, 1H, 2J = 10.0 Hz), 2.94 (d, 1H, 2J = 10.0
Hz), 2.73 (m, 2H), 2.53 (ddd, 1H, 2J = 14.0 Hz, 3J = 6.5 and 4.5 Hz),
2.43 (m, 1H), 1.55 (s, 3H), 1.25−1.21 (m, 12H). 13C NMR (125
MHz, CD3CN) δ: 163.7, 158.9, 150.4, 144.5, 137.6 (q, J = 39 Hz),
135.8, 135.1, 134.9, 130.1, 128.0, 127.1, 125.9, 120.9 (q, J = 265 Hz),
118.6, 113.2, 110.6, 87.0, (85.4 and 85.3), 84.0, (74.7 and 74.6), 63.3,
(58.5 and 58.3), 54.9, 51.9, (43.3 and 43.2), 38.3, (24.3 and 24.2),
(23.91 and 23.85), (20.2 and 20.1), 11.3. 31P NMR (200 MHz,
CD3CN) δ: 149.7. 19F NMR (470 MHz, CD3CN) δ: −63.28. HRMS
(ESI-TOF) m/z: calcd for C44H51F3N7NaO8P [M + Na]+ 916.3387,
found 916.3387.
Oligonucleotide Synthesis. Oligonucleotides were synthesized
on a 1.0 μmol scale using an automatic DNA/RNA synthesizer.
Benzylthiotetrazole as an activator and coupling times of 20 and 300 s
were used for the couplings of standard DNA and RNA building
blocks, respectively. Automatic coupling of 12: A 0.13 mol L−1 solution
of 12 was prepared and added to the synthesizer. The coupling time
was increased to 600 s, and otherwise the standard coupling cycle was
used. According to the DMTr assay, a 92% coupling efficiency was
obtained. Manual coupling of 12: A 0.20 mol L−1 solution of 12 (10
μmol) was prepared. This solution and a solution of benzylthiote-
trazole (0.25 mol L−1 in dry acetonitrile, 10 μmol) were suspended
with the CPG support (bearing the sequence before 12, 1 μmol). The
suspension was mixed for 10 min under nitrogen at ambient
temperature, loaded onto the synthesis column, and filtered. The
synthesis column was set to the synthesizer, and then the chain
elongation was continued automatically. According to the DMTr
3535
dx.doi.org/10.1021/jo500326j | J. Org. Chem. 2014, 79, 3529−3536

The Journal of Organic Chemistry
Article
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2535.
ASSOCIATED CONTENT
* Supporting Information
NMR spectra for 11 and 12, RP HPLC profiles and MS (ESI-
TOF) data for ON1 and the labeled HIV-1 TAR models, and
UV and CD data for the oligonucleotides. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
(31) Thrane, H.; Fensholdt, J.; Regner, M.; Wengel, J. Tetrahedron
1995, 51, 10389.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: pamavi@utu.fi.
■
6449.
(33) Kiviniemi, A.; Virta, P.; Lonnberg, H. Bioconjugate Chem. 2008,
19, 1726.
̈
Notes
The authors declare no competing financial interest.
(34) Kiviniemi, A.; Virta, P.; Lonnberg, H. Bioconjugate Chem. 2010,
̈
21, 1890.
ACKNOWLEDGMENTS
■
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Org. Chem. 2001, 3013.
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Financial support from the Academy of Finland (251539 and
256214) is gratefully acknowledged. We also thank Anu
Kiviniemi for some preliminary work considered in this study.
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